PROJECT SUMMARY The hematopoietic system follows a hierarchical organization, with multipotent long-term repopulating hematopoietic stem cells (LT-HSCs) occupying the top tier. This paradigm, developed mostly through cell transplantation assays, has recently been contested by a series of studies performed under native conditions, without transplantation. Application of systems-level single cell methods in this setting has revealed a heterogeneity of cell states within progenitors and stem cells, prompting a reevaluation of the theories of hematopoietic lineage fate decisions. As a research fellow, I described that hematopoietic stem cell fates are clonally heterogeneous under steady state and follow a complex landscape of lineage fate choices. In addition, my work uncovered that a fraction of LT-HSCs contributes to a significant proportion of the megakaryocytic cell lineage under steady state while rarely generating other types of progeny in unperturbed conditions. For my transition to independence, my objective will be to describe the molecular underpinnings of blood stem cell heterogeneity, with a special focus on clonal expansion and lineage bias, which are prevalent phenotypes of aging HSCs and are related with severe diseases. I have recently developed two novel systems that allow native barcoding hematopoietic cells at the RNA level in order to simultaneously capture the cell fates and transcriptional states of HSCs and will use them to define cell-state maps pre- and post- transplantation (Aim 1a). In my independent phase, I will also establish a model for performing cell barcoding in humanized HSC models and develop steady state humanized progenitor state-fate maps (Aim 1b). From the analysis of preliminary state-fate maps, I have designed and carried out a CRISPR-based screening for HSC state-fate regulators, and discovered novel gene candidates that control HSC heterogeneity. In my second aim, I will individually characterize selected candidate mechanisms, and explain how they contribute to unique HSC functions (Aim 2a). Finally, the machineries controlling HSC fates have never been addressed using specific gene interference in the LT-HSC compartment and I have devised a new method to perform HSC-specific gene silencing in mice (Aim 2b). The mentored phase of this award will take place at the Stem Cell Program at Boston Children's Hospital (BCH). There, I will be guided by two internationally acclaimed leaders in the field of hematopoiesis, Dr. Fernando Camargo and Dr. Leonard Zon (Program Director). I will be further advised by a scientific committee of experts covering different aspects of my project: David Scadden, George Church and Allon Klein. Together, we've developed a comprehensive scientific and career development plan that will provide me with the necessary skills to transition to independence by the end of this program. My career goal is to use clonal in situ lineage tracing technologies to dissect adult stem cell heterogeneity and its relationship to the origin of important blood disorders, and facilitate the translation of drug-based and stem cell-based therapies.